CN117625541A - Brain glioma organoid construction method and drug sensitivity detection method - Google Patents
Brain glioma organoid construction method and drug sensitivity detection method Download PDFInfo
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Abstract
The invention discloses a brain glioma organoid construction method and a drug sensitivity detection method, which relate to the technical field of brain glioma organoids and solve the problems that the construction quality of the brain glioma organoids is insufficient and the drug sensitivity detection of the brain glioma organoids cannot be realized, wherein a bracket module, a bracket surface modification module and a cell three-dimensional culture module are prepared to realize the accurate control of the cell three-dimensional culture environment, the construction quality of the brain glioma organoids is improved, the mechanical stress test and the chemical corrosion test are used for carrying out the mechanical property test and the corrosion resistance test on the three-dimensional biological bracket of the brain glioma organoids, a matrix gel wrapping cell detection module, a chemiluminescence method detection module and an ATP adenosine triphosphate quantitative analysis detection module are used for evaluating the biological characteristics and the efficacy of the brain glioma organoids, and nuclear magnetic resonance detection method and a cell profiler statistical software are used for determining the influence of selected drugs on the growth or metabolism of cells so as to realize the drug sensitivity detection of the brain glioma organoids.
Description
Technical Field
The invention relates to the technical field of brain glioma organoids, in particular to a brain glioma organoid construction method and a drug sensitivity detection method.
Background
The main function of the brain glioma organoid construction method and the drug sensitivity detection method is to evaluate the inhibition effect of different chemical drugs on brain glioma cells in vitro, and provide guidance for clinical treatment schemes. The principle is that an organoid model for highly reducing the tumor tissue morphology and microenvironment of a patient is prepared based on a three-dimensional printing technology, chemical drugs to be detected are added into a culture solution, and the inhibition effect of different chemical drugs on the growth and proliferation of tumor cells is evaluated in the organoid model. By comparing the inhibition effect of different drugs on tumor cells, the sensitivity of patients to different chemical drugs can be predicted, thereby providing reference for individuation treatment.
In the prior art, a brain glioma organoid construction method and a drug sensitivity detection method have a plurality of defects, on one hand, the three-dimensional culture environment of cells cannot be accurately controlled, so that the effect of a brain glioma organoid model which cultures the cells into a three-dimensional structure is insufficient, the three-dimensional biological stent of the brain glioma organoid model is lack of mechanical property test and corrosion resistance test, the construction quality of the brain glioma organoid model is influenced, on the other hand, the biological characteristics and the efficacy of the brain glioma organoid model cannot be evaluated, so that the constructed brain glioma organoid model does not have biological characteristics and functions, and the drug sensitivity detection method of the brain glioma organoid model cannot be known, therefore, the invention provides the brain glioma organoid construction method and the drug sensitivity detection method, and aims at improving the construction quality of the brain glioma organoid model and realizing the drug sensitivity detection of the brain glioma organoid.
Disclosure of Invention
In order to overcome the defects of the technology, the invention discloses a brain glioma organoid construction method and a drug sensitivity detection method, wherein cells are planted on a three-dimensional biological scaffold by preparing a scaffold module, a scaffold surface modification module and a cell three-dimensional culture module, the cells are cultured into a brain glioma organoid model with a three-dimensional structure, the problem that the effect of the brain glioma organoid model with the three-dimensional structure is insufficient due to the fact that the three-dimensional culture environment of the cells cannot be accurately controlled is solved, the mechanical property test and the corrosion resistance test are carried out on the three-dimensional biological scaffold of the brain glioma organoid model by the chemical abrasion test through the mechanical stress test and the chemical corrosion test, the problem that the mechanical property test and the corrosion resistance test are carried out on the three-dimensional biological scaffold of the brain glioma organoid model are lack, the biological property and the efficacy of the brain glioma organoid model are evaluated by a matrix gel wrapping cell detection module, a chemiluminescent method detection module and an ATP triphosphates quantitative analysis detection module, the biological property and the efficacy of the brain glioma organoid model cannot be evaluated, the problem that the constructed brain glioma organoid model does not have the biological property and the function is solved, the problem that the drug sensitivity of the brain glioma organoid model cannot be solved by the detection method and the cell has no effect on the drug sensitivity test on the cell tumor organoid is realized by the cell resonator.
Analysis in view of the above, the present invention provides a method for constructing a brain glioma organoid, the method comprising the steps of:
firstly, separating cells from a glioma sample by primary separation of tissue cells, and inoculating and culturing the cells according to a cell counting result;
step two, planting cells on the three-dimensional biological scaffold by adopting a three-dimensional cell culture method, and culturing the cells into a brain glioma organoid model with a three-dimensional structure, wherein the three-dimensional cell culture method comprises the steps of preparing a scaffold module, a scaffold surface modification module and a cell three-dimensional culture module;
in the second step, the output end of the preparation bracket module is connected with the input end of the bracket surface modification module, and the output end of the bracket surface modification module is connected with the input end of the cell three-dimensional culture module;
step three, performing mechanical property test and corrosion resistance test on the three-dimensional biological scaffold of the brain glioma organoid model by adopting chemical abrasion test, wherein the chemical abrasion test comprises mechanical stress test and chemical corrosion test;
step four, biological characteristics and efficacy of a brain glioma organoid model are evaluated through a chemical and physical detection method, wherein the chemical and physical detection method comprises a matrigel coated cell detection module, a chemiluminescence method detection module and an ATP adenosine triphosphate quantitative analysis detection module;
In the fourth step, the output end of the matrigel coated cell detection module is connected with the input end of the chemiluminescence method detection module, and the output end of the chemiluminescence method detection module is connected with the input end of the ATP adenosine triphosphate quantitative analysis detection module;
fifthly, adding the selected medicine into a culture plate with a brain glioma organoid model, determining the influence of the selected medicine on the growth or metabolism of cells through a nuclear magnetic resonance detection method and a cell profiler statistical software, and realizing the drug sensitivity detection of the brain glioma organoid model, wherein the selected medicine at least comprises temozolomide, methylbenzyl hydrazine, lomustine, vincristine, carmustine, etoposide, irinotecan, cisplatin, carboplatin and bevacizumab.
As a further technical scheme of the invention, the preparation stent module adopts matrigel as a three-dimensional biological stent material, the stent surface modification module forms a silicon dioxide layer on the matrigel three-dimensional biological stent through trichloromethylsilane, the mechanical strength of the matrigel three-dimensional biological stent is improved to be 6.5 MPa, and the stent surface modification module forms a pore diameter of 150 on the surface of the matrigel three-dimensional biological stent through ion implantation treatment Is provided to enhance cell adhesion.
As a means ofAccording to the further technical scheme, the cell three-dimensional culture module enables cells to invade the inside of the matrigel three-dimensional biological scaffold by immersing the matrigel three-dimensional biological scaffold in the cell culture solution, and the cell three-dimensional culture module controls the pressure, the temperature and the temperature of the cell culture solution by adopting a model predictive control algorithmThe concentration is used for realizing accurate control of the three-dimensional cell culture environment, and the working method of the model predictive control algorithm comprises the following steps:
step one, establishing the pressure, the temperature and the culture solution by adopting a state space equationDynamic equations between concentrations, which are used to describe pressure, temperature and +.>The model predictive control algorithm is based on algebraic equation set theory to control the pressure, temperature and +.>The concentration is used as an unknown variable, a culture solution dynamic model is constructed by utilizing a reaction rate constant and a growth rate, and the calculation formula of the dynamic equation is as follows:
（1）
in the case of the formula (1),for dynamic equation>For the pressure of the culture broth, < > is given>For the temperature of the culture solution, < > is->Is a culture solutionConcentration of->A coefficient matrix which is a state space equation;
step two, the culture solution dynamic model regards the three-dimensional cell culture environment as a space grid consisting of cells through a system dynamics method, and each cell comprises 1 The nutrient, the dynamic model of said culture solution predicts the condition of the future change of pressure, temperature, carbon dioxide of the culture solution on the basis of the interaction relation and cell behavior law among cell, the computational formula of the dynamic model of said culture solution predicts the pressure variation of the culture solution is:
（2）
in the formula (2) of the present invention,predicting the pressure variation of the culture solution for the culture solution dynamic model,/->For cell adhesion between the cells +.>Predicting the current pressure value of the culture solution for the culture solution dynamic model,/->Is the cell migration velocity;
the temperature change amount calculation formula of the culture solution dynamic model prediction culture solution is as follows:
（3）
in the formula (3) of the present invention,predicting the temperature variation of the culture solution for the culture solution dynamic model,/->For the heating power of the heater, +.>For cultivation time, ->Heat conduction rate for three-dimensional cell culture environment, +.>The cell three-dimensional culture environment capacity is used for cells;
the calculation formula for predicting the carbon dioxide concentration variation of the culture solution by the culture solution dynamic model is as follows:
（4）
in the formula (4) of the present invention,predicting the carbon dioxide concentration variation of the culture solution for the culture solution dynamic model,/->For cell growth rate, ++>Supplying system power for carbon dioxide, < >>Rate of production of cellular metabolites;
Step three, a main controller is adopted to output control signals in real time according to the prediction result of the culture solution dynamic model and the target set value of the three-dimensional cell culture environment, and the model prediction control algorithm adjusts the power of the heater and the opening of the exhaust valve according to the control signalsAnd a carbon dioxide supply system for supplying pressure, temperature andthe concentration is kept at a target set value, and the calculation formula of the control signal is as follows:
（5）
in the formula (5) of the present invention,for control signal +.>Proportional gain for model predictive control algorithm, +.>Outputting control signal time for the main controller, +.>To limit the saturation clipping of the size range of the output control signal.
According to the technical scheme, the hydraulic system is used for vibrating or loading the three-dimensional biological support of the brain glioma organoid model, the use condition of the three-dimensional biological support by mechanical stress in the internal environment of a human body is simulated, the hydraulic system is used for setting the pressure and flow of the hydraulic cylinder according to the test requirement by adopting a PID control algorithm, so that the hydraulic cylinder converts liquid into mechanical motion to act on the three-dimensional biological support sample of the brain glioma organoid model, the hydraulic system is used for acquiring test data of the load and deformation of the three-dimensional biological support of the brain glioma organoid model in the test process by using a sensor, the hydraulic system is used for analyzing and processing the acquired test data by using LabVIEW data analysis software, and the LabVIEW data analysis software is used for comparing the test data with a theoretical model by using a curve fitting function, so that the yield strength performance of the three-dimensional biological support is obtained.
As a further technical scheme of the invention, the chemical corrosion test adopts a sodium carbonate-potassium hydroxide buffer solution to simulate the degradation condition of the three-dimensional biological stent in the internal environment of a human body, and after the three-dimensional biological stent sample of the glioma organoid model is soaked in the sodium carbonate-potassium hydroxide buffer solution for 7 days, the chemical corrosion test adopts a scanning electron microscope to compare the appearance, quality and chemical composition of the sample before and after the test, so as to evaluate the corrosion resistance of the three-dimensional biological stent of the glioma organoid model in the internal environment of the simulated human body.
As a further technical scheme of the invention, the matrigel coated cell detection module utilizes matrigel to simulate the in-vivo environment of brain glioma organoid cells, the proliferation condition of the brain glioma organoid cells in a three-dimensional space is observed and analyzed, the matrigel coated cell detection module adopts a flow cell detection method to detect matrigel coated with fluorescein PI to simulate brain glioma organoid cell samples, the flow cell detection method determines the DNA content contained in each cell by detecting the fluorescence intensity of the fluorescein PI, the flow cell detection method distinguishes cells in different periods according to a DNA content scatter diagram, the proportion of cells in different periods in the samples is determined, the cell period comprises four stages of G1 phase, S phase, G2 phase and M phase, and finally the flow cell detection method calculates the cell proliferation rate and the period analysis result according to the proportion of cells in each period.
As a further technical scheme of the invention, the chemiluminescent method detection module adopts coupled luciferase to dye to realize the selective marking of apoptotic cells, the apoptotic cells generate luminescent signals after being catalyzed by the coupled luciferase, and the chemiluminescent method detection module uses a fluorescence microscope and a filter to observe and screen the emission spectrum, so as to obtain high selectivity and sensitivity of the apoptotic cells and realize the detection of apoptosis conditions.
According to the invention, the ATP triphosadenine quantitative analysis and detection module is mixed with the brain glioma organoid model, the mixed solution of the trichloroacetic acid and the brain glioma organoid model breaks tissue cells in the brain glioma organoid model through ultrasonic mechanical breaking and releases ATP triphosadenine, the ATP triphosadenine quantitative analysis and detection module is used for centrifuging the mixed solution after breaking the tissue by adopting a centrifuge, an ATPIase inhibitor and a chloroform organic solvent, preventing ATP triphosadenine from degrading and separating ATP triphosadenine in supernatant, the ATP triphosadenine quantitative analysis and detection module is used for generating a product capable of emitting fluorescent signals through the mixed reaction of fluorescein, hydrogen peroxide and luciferase catalyst and the separated ATP triphosadenine, and finally the ATP triphosadenine quantitative analysis and detection module is used for quantitatively calculating the ATP content according to the fluorescent intensity by observing and analyzing the fluorescent signals generated by a fluorescent detector, so that the activity of the brain glioma organoid model is measured and the convenience and the accuracy of the brain glioma organoid sensitization detection are improved.
According to the nuclear magnetic resonance detection method, a centrifugal machine, a filtering membrane and a PBS buffer solution are adopted to pretreat a cell culture solution, bubbles and particles in the cell culture solution are removed, a nuclear magnetic resonance instrument is used for generating specific nuclear magnetic resonance signals by using metabolites and compounds in the cell culture solution, signals of different molecules in the cell culture solution are detected, nuclear magnetic resonance spectrum analysis is carried out on the signals of the molecules through statistical software of the cytoprofiler, the proportion of the metabolites and the compounds in a sample is determined, and finally the nuclear magnetic resonance detection method is used for indicating the influence of the selected drugs on the growth or metabolism of the cells according to the proportion of the metabolites and the compounds in the sample.
As a further technical scheme of the invention, the drug sensitivity detection method for the brain glioma organoids is applied to the construction method for the brain glioma organoids, and comprises the following steps:
transferring a brain glioma organoid culture plate into a biosafety cabinet, transferring the brain glioma organoid in a suction hole of a pipetting gun into a sterile 15mL centrifuge tube, repeatedly blowing, centrifuging in a centrifuge at the temperature of-4 ℃ to 4 ℃, discarding supernatant to leave a precipitate, re-suspending by adopting a culture medium according to the volume of the precipitate, taking an equal volume of cell suspension and trypan blue solution, fully and uniformly mixing the cell suspension and the trypan blue solution according to the ratio of 1:1, counting, and performing drug sensitive plating and culture according to a drug recommendation scheme;
Dividing the brain glioma organoid model into a drug treatment group and a control group of temozolomide, methylbenzyl hydrazine, lomustine, vincristine, carmustine, etoposide, irinotecan, cisplatin, carboplatin and bevacizumab;
step three, respectively adding temozolomide, methylbenzyl hydrazine, lomustine, vincristine, carmustine, etoposide, irinotecan, cisplatin, carboplatin and bevacizumab into each drug treatment group, and gradually increasing the concentrations of 0.1g/ml, 0.2g/ml, 0.3g/ml, 0.4g/ml and 0.5g/ml to obtain drug concentration-effect relation data, wherein no drug is added into the control group, and the culture plates of the drug treatment group and the control group are placed into a culture box for culture;
step four, adding an equivalent amount of ATP luminescent detection reagent into culture plate holes of each drug treatment group and each control group, detecting by adopting an enzyme-labeled instrument with a luminescent detection function, processing and analyzing cell values in each sample by adopting a nuclear magnetic resonance detection method and cell profilometer statistical software, and detecting the influence of drugs on the growth or metabolism of cells;
and fifthly, correspondingly comparing the cell growth or metabolism conditions in the drug treatment group and the control group to finally obtain the drug sensitivity of temozolomide, methylbenzyl hydrazine, lomustine, vincristine, carmustine, etoposide, irinotecan, cisplatin, carboplatin and bevacizumab to brain glioma organoids.
The invention has positive and beneficial effects different from the prior art:
the invention discloses a brain glioma organoid construction method and a drug sensitivity detection method, wherein cells are planted on a three-dimensional biological scaffold by preparing a scaffold module, a scaffold surface modification module and a cell three-dimensional culture module, the cells are cultured into a brain glioma organoid model with a three-dimensional structure, the three-dimensional cell culture environment is accurately controlled, the construction quality of the brain glioma organoid model with the three-dimensional structure is improved, a mechanical stress test and a chemical corrosion test are carried out on the three-dimensional biological scaffold of the brain glioma organoid model by a chemical abrasion test, a mechanical property test and a corrosion resistance test are carried out on the three-dimensional biological scaffold of the brain glioma organoid model by a chemical and physical detection method, the biological characteristics and the efficacy of the brain glioma organoid model are evaluated by a matrigel encapsulation cell detection module, a chemiluminescence method detection module and an ATP triphosadenine quantitative analysis detection module, the influence of the selected drugs on the growth or metabolism of the cells is ensured to be determined by a resonance detection method and a cell profilometer statistical software, and the drug sensitivity detection of the brain glioma organoid model is realized.
Drawings
For a clearer description of embodiments of the invention or of solutions in the prior art, the drawings that are necessary for the description of the embodiments or of the prior art will be briefly described, it being apparent that the drawings in the description below are only some embodiments of the invention, from which, without inventive faculty, other drawings can be obtained for a person skilled in the art, in which:
FIG. 1 is a general flow chart of a method of constructing a brain glioma organoid according to the present invention;
FIG. 2 is a general flow chart of a method for detecting drug sensitivity of brain glioma organoids according to the present invention;
FIG. 3 is a schematic diagram of a model predictive control algorithm architecture employed in the present invention;
FIG. 4 is a schematic diagram of a three-dimensional cell culture method according to the present invention;
FIG. 5 is a schematic diagram of the chemical and physical detection method architecture employed in the present invention.
Detailed Description
The following description of the embodiments of the present disclosure will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the disclosure. It should be understood that the description is only illustrative and is not intended to limit the scope of the invention. In addition, in the following description, descriptions of well-known structures and techniques are omitted so as not to unnecessarily obscure the present invention.
As shown in fig. 1 to 5, a method for constructing a brain glioma organoid, the method comprising the steps of:
firstly, separating cells from a glioma sample by primary separation of tissue cells, and inoculating and culturing the cells according to a cell counting result;
step two, planting cells on the three-dimensional biological scaffold by adopting a three-dimensional cell culture method, and culturing the cells into a brain glioma organoid model with a three-dimensional structure, wherein the three-dimensional cell culture method comprises the steps of preparing a scaffold module, a scaffold surface modification module and a cell three-dimensional culture module;
in the second step, the output end of the preparation bracket module is connected with the input end of the bracket surface modification module, and the output end of the bracket surface modification module is connected with the input end of the cell three-dimensional culture module;
step three, performing mechanical property test and corrosion resistance test on the three-dimensional biological scaffold of the brain glioma organoid model by adopting chemical abrasion test, wherein the chemical abrasion test comprises mechanical stress test and chemical corrosion test;
step four, biological characteristics and efficacy of a brain glioma organoid model are evaluated through a chemical and physical detection method, wherein the chemical and physical detection method comprises a matrigel coated cell detection module, a chemiluminescence method detection module and an ATP adenosine triphosphate quantitative analysis detection module;
In the fourth step, the output end of the matrigel coated cell detection module is connected with the input end of the chemiluminescence method detection module, and the output end of the chemiluminescence method detection module is connected with the input end of the ATP adenosine triphosphate quantitative analysis detection module;
fifthly, adding the selected medicine into a culture plate with a brain glioma organoid model, determining the influence of the selected medicine on the growth or metabolism of cells through a nuclear magnetic resonance detection method and a cell profiler statistical software, and realizing the drug sensitivity detection of the brain glioma organoid model, wherein the selected medicine at least comprises temozolomide, methylbenzyl hydrazine, lomustine, vincristine, carmustine, etoposide, irinotecan, cisplatin, carboplatin and bevacizumab.
In a specific embodiment, the brain glioma-like vessel construction operation flow is as follows: after receiving the sample, discarding the tissue preservation solution in the sample tube in the biosafety cabinet, cleaning the tissue for a plurality of times by using the cleaning solution, and discarding the cleaning solution. The tissue samples were then transferred to sterile EP tubes and minced tissue was sheared using sterile surgical scissors and forceps. Then transferring the sheared tissues into a centrifuge tube, adding a proper amount of tissue digestion liquid, uniformly mixing, and placing in a water bath kettle at 37 ℃ for shaking digestion. After completion of digestion, the stop solution was added to terminate digestion and filtration was performed. The filtrate was centrifuged in a low temperature centrifuge and the supernatant discarded leaving a precipitate. Resuspension was performed with an appropriate amount of medium according to the volume of the pellet, and equal volumes of cell suspension and trypan blue solution were counted after thoroughly mixing at 1:1. According to the cell count result, inoculating culture is carried out.
In a further embodiment, the preparation stent module adopts matrigel as a three-dimensional biological stent material, the stent surface modification module forms a silicon dioxide layer on the matrigel three-dimensional biological stent through trichloromethylsilane, the mechanical strength of the matrigel three-dimensional biological stent is improved to be 6.5 MPa, and the stent surface modification module forms a pore diameter of 150 on the surface of the matrigel three-dimensional biological stent through ion implantation treatmentIs provided to enhance cell adhesion.
In particular embodiments, the DMEM medium is a widely used cell culture medium containing high nutrients that can support the growth and proliferation of many different types of mammalian cells. The function and principle are as follows: providing nutrients: DMEM medium contains a variety of essential nutrients including amino acids, glucose, nucleic acids, lipids, etc., which are necessary for cell growth and metabolism. In DMEM medium, cells can grow and proliferate by absorbing these nutrients. Maintaining the pH value stable: the DMEM medium contains a buffer (e.g., HEPES) and the pH of the culture medium can be adjusted to maintain the pH within a suitable range. This prevents damage to the cells at too high or too low a pH. Providing a serum fraction: DMEM medium typically requires the addition of 10% Fetal Bovine Serum (FBS), which contains various growth factors, hormones and other components that promote cell growth and proliferation and provide antibodies, iron ions, etc. that are essential for certain specific cell types. Inhibiting cell infection, and preventing proliferation of cells due to bacterial and fungal contamination by antibiotics (such as penicillin/streptomycin) contained in DMEM medium. In summary, DMEM media provides nutrients, buffers and other components necessary for growth, providing a suitable environment for cell growth in vitro.
Preparing a bracket module, a bracket surface modification module and a cell three-dimensional culture module, and planting cells on the three-dimensional biological bracket to prepare the brain glioma organoid model. The function of these modules is as follows: the scaffold module is an integral part for constructing a three-dimensional biological scaffold. They provide a structure made of porous material that provides cells with a support and orientation similar to the in vivo environment, promoting cell-cell interactions and tissue formation. Stent surface modification modules are a technique for altering the surface properties of three-dimensional biological stents by altering the chemical and physical properties of the material surface to regulate its interaction with the surrounding environment or cells planted thereon. For example, by varying the parameters of surface charge, chemical functionality, etc., the adhesion between the scaffold and the surrounding environment or tissue on which it is grown, mobility, etc., can be controlled. When cells are planted on a three-dimensional biological scaffold, the cells need to be cultured into a three-dimensional structure. A cell three-dimensional culture module is a technique for culturing and assembling cells on a three-dimensional biological scaffold. By the technology, the distribution of cells in the stent, proliferation rate, constitutive tissue structure and other parameters can be controlled, so that the formation of the organ structure is realized. In summary, the scaffold modules, scaffold surface modification modules, and cell three-dimensional culture modules are prepared in order to plant and culture specific types of cells on a three-dimensional biological scaffold and to form a tissue structure having an in vivo-like environment. The techniques have important application values in the fields of researching disease occurrence mechanisms, screening medicines and tissue engineering.
In a further embodiment, the cell three-dimensional culture module enables cells to invade the inside of the matrigel three-dimensional biological scaffold by immersing the matrigel three-dimensional biological scaffold in a cell culture solution, and the cell three-dimensional culture module controls the pressure, the temperature and the cell culture solution by adopting a model predictive control algorithmThe concentration is used for realizing accurate control of the three-dimensional cell culture environment, and the working method of the model predictive control algorithm comprises the following steps:
step one, establishing the pressure, the temperature and the culture solution by adopting a state space equationDynamic equations between concentrations, which are used to describe pressure, temperature and +.>The model predictive control algorithm is based on algebraic equation set theory to control the pressure, temperature and +.>The concentration is used as an unknown variable, a culture solution dynamic model is constructed by utilizing a reaction rate constant and a growth rate, and the calculation formula of the dynamic equation is as follows:
（1）
in the case of the formula (1),for dynamic equation>For the pressure of the culture broth, < > is given>For the temperature of the culture solution, < > is->Is a culture solutionConcentration of->A coefficient matrix which is a state space equation;
step two, the culture solution dynamic model regards the three-dimensional cell culture environment as a space grid consisting of cells through a system dynamics method, and each cell comprises 1 The nutrient, the dynamic model of said culture solution predicts the condition of the future change of pressure, temperature, carbon dioxide of the culture solution on the basis of the interaction relation and cell behavior law among cell, the computational formula of the dynamic model of said culture solution predicts the pressure variation of the culture solution is:
（2）
in the formula (2) of the present invention,predicting the pressure variation of the culture solution for the culture solution dynamic model,/->For cell adhesion between the cells +.>Predicting the current pressure value of the culture solution for the culture solution dynamic model,/->Is the cell migration velocity;
the temperature change amount calculation formula of the culture solution dynamic model prediction culture solution is as follows:
（3）
in the formula (3) of the present invention,predicting the temperature variation of the culture solution for the culture solution dynamic model,/->For the heating power of the heater, +.>For cultivation time, ->Heat conduction rate for three-dimensional cell culture environment, +.>The cell three-dimensional culture environment capacity is used for cells;
the calculation formula for predicting the carbon dioxide concentration variation of the culture solution by the culture solution dynamic model is as follows:
（4）
in the formula (4) of the present invention,predicting the carbon dioxide concentration variation of the culture solution for the culture solution dynamic model,/->For cell growth rate, ++>Supplying system power for carbon dioxide, < >>Rate of production of cellular metabolites;
Step three, a main controller is adopted to output control signals in real time according to the prediction result of the culture solution dynamic model and the target set value of the three-dimensional cell culture environment, and the model prediction control algorithm is controlled according to the controlSignals adjust heater power, exhaust valve opening and carbon dioxide supply system to pressure, temperature andthe concentration is kept at a target set value, and the calculation formula of the control signal is as follows:
（5）
in the formula (5) of the present invention,for control signal +.>Proportional gain for model predictive control algorithm, +.>Outputting control signal time for the main controller, +.>To limit the saturation clipping of the size range of the output control signal.
In a specific embodiment, an application platform of the model predictive control algorithm may be constructed, and during the working process of the platform, a hardware platform may be constructed, for example, by constructing the following components for processing, for example: the following is a more detailed description of the embodiments:
a sensor: for collecting environmental or system status data such as temperature, humidity, pressure, etc.;
a data acquisition card: converting the data collected by the sensor into digital signals, and processing and storing the digital signals;
And (3) a controller: calculating a control strategy by using a model predictive control algorithm, and converting the control strategy into an executable command to adjust the system state;
and a communication module: the method is used for data interaction and control instruction transmission between different components. For example, the respective devices are connected using a wireless network or a wired network;
and (3) power supply: providing necessary power support for all components;
in a word, when the model predictive control algorithm application platform is constructed, proper hardware components are required to be selected according to specific scenes and requirements, and reasonable combination and configuration are performed so as to ensure that the system operates normally and can effectively realize required functions.
The model predictive control algorithm (Model Predictive Control, MPC) is a control method based on a dynamic mathematical model, and the main idea is to optimize the control strategy at the current moment through predicting the future state of the system so as to realize the accurate control of the state variables of the system. In the cell three-dimensional culture module, the MPC algorithm can predict the change trend of environmental factors such as culture fluid pressure, temperature, concentration and the like in a period of time in the future according to the existing dynamic model and real-time measurement data, and calculate an optimal control strategy so as to keep the environmental parameters to stably run in a target range. Specifically, at each sampling time point, the MPC algorithm uses the state at the current time as an initial condition, performs multi-step prediction using a system dynamic model, and calculates a set of optimal control input sequences. Then, the first control input is performed in the next sampling period and the state estimation and prediction is repeated. The process is repeated until the termination condition is met or the system stops working, a model predictive control algorithm (Model Predictive Control, MPC) adopts a main controller to output control signals in real time according to the dynamic model predictive result of the culture solution and the target set value of the three-dimensional culture environment of the cells, and the main function is to precisely control the three-dimensional culture environment of the cells to be always in a stable state, so that the quality and the yield of the cell culture are improved, and particularly, the MPC algorithm calculates the optimal control input sequence by predicting the future state of the system and combining the information of the target set value, the constraint condition and the like. These control input sequences include strategies to regulate factors such as culture fluid pressure, temperature, and concentration, and are continually updated and adjusted during real-time operation. In this way, the environmental parameters can be kept to stably run within the target range, abnormal cell growth or death caused by excessively or insufficiently adjusting the environmental parameters can be avoided, and in addition, the MPC algorithm can also predict and timely correct possible abnormal conditions. For example, when a system fails or an emergency occurs, the MPC algorithm can be adjusted accordingly according to the existing model prediction results, and the influence on the cell growth is reduced as much as possible. Therefore, the MPC algorithm plays an important role in the control of the three-dimensional cell culture environment, and can effectively improve the production efficiency and quality. The statistical table of the calculation results of the control signals is shown in table 1:
Table 1 calculation result statistics table of control signals
As shown in Table 1, four test groups are set, control signals are calculated by two methods, a statistical analysis method is adopted to infer calculation control signals for historical data or sample data by the method 1, a model prediction control algorithm is adopted to output control signals in real time by a main controller according to a culture solution dynamic model prediction result and a target set value of a cell three-dimensional culture environment by the method 2, the error of the method 1 is larger than that of the method 2, and the model prediction control algorithm has outstanding technical effects by adopting the main controller to output control signals in real time according to the culture solution dynamic model prediction result and the target set value of the cell three-dimensional culture environment by the method 1.
In a further embodiment, the mechanical stress test adopts a hydraulic system to vibrate or load the three-dimensional biological scaffold of the brain glioma organoid model, the use condition of the mechanical stress in the internal environment of a human body to the three-dimensional biological scaffold is simulated, the hydraulic system adopts a PID control algorithm to set the pressure and flow of a hydraulic cylinder according to the test requirement, the hydraulic system converts liquid into mechanical motion to act on the three-dimensional biological scaffold sample of the brain glioma organoid model, the hydraulic system acquires test data of the three-dimensional biological scaffold load and deformation of the brain glioma organoid model in the test process through a sensor, the hydraulic system adopts LabVIEW data analysis software to analyze and process the acquired test data, and the LabVIEW data analysis software adopts a curve fitting function to compare the test data with a theoretical model, so as to acquire the yield strength performance of the three-dimensional biological scaffold.
In a further embodiment, the chemical corrosion test adopts a sodium carbonate-potassium hydroxide buffer solution to simulate the degradation condition of the three-dimensional biological scaffold in the internal environment of a human body, and after the three-dimensional biological scaffold sample of the glioma organoid model is soaked in the sodium carbonate-potassium hydroxide buffer solution for 7 days, the chemical corrosion test adopts a scanning electron microscope to compare the appearance, quality and chemical composition of the sample before and after the test, so as to evaluate the corrosion resistance of the three-dimensional biological scaffold of the glioma organoid model in the internal environment of the simulated human body.
In a specific embodiment, the mechanical stress test is a test method for simulating the mechanical stress in the internal environment of a human body by vibrating or loading a three-dimensional biological scaffold of a brain glioma organoid model so as to obtain the yield strength performance of the brain glioma organoid model, and in the test, a hydraulic system can apply a certain load or vibration to the three-dimensional biological scaffold so as to simulate the mechanical stress in the internal environment of the human body. The load or vibration can deform the three-dimensional biological scaffold, so that corresponding data such as reaction force and displacement are generated. From these data, the yield strength performance parameters of the three-dimensional biological scaffold can be obtained through calculation and analysis, specifically, the yield strength refers to the maximum stress value born by the material when plastic deformation starts to occur after the material is subjected to a certain load. The method for testing the mechanical stress of the three-dimensional biological scaffold by using the hydraulic system is a method for effectively evaluating the yield strength performance of the three-dimensional biological scaffold, and has important significance for designing and optimizing the scaffold.
The chemical corrosion test is a test method for evaluating the corrosion resistance of a three-dimensional bioscaffold of a brain glioma organoid model in a simulated human internal environment by simulating the condition in the human internal environment using a sodium carbonate-potassium hydroxide buffer solution, in which the three-dimensional bioscaffold is immersed in the sodium carbonate-potassium hydroxide buffer solution to simulate the chemical environment in the human internal environment. The buffer solution can stabilize the pH value to be close to the pH value of human tissues and blood, can provide necessary ions and nutrient substances, observe parameters such as surface change, weight loss and the like of the three-dimensional biological scaffold within a certain time, and evaluate the corrosion resistance according to the data. For example, if the three-dimensional biological stent has obvious degradation, rough surface, increased weight loss and the like, the corrosion resistance is poor; on the contrary, the corrosion resistance is better, and it is noted that when the chemical corrosion test is performed, the test conditions such as temperature, buffer concentration, soaking time and the like are controlled, so as to ensure the reliability and accuracy of the test, in a word, the sodium carbonate-potassium hydroxide buffer solution is adopted to simulate the degradation condition of the three-dimensional biological scaffold in the internal environment of the human body, and the corrosion resistance is evaluated as an effective method. The test method can provide reference basis for the design and optimization of the three-dimensional biological scaffold, and is beneficial to improving the stability and safety of the three-dimensional biological scaffold in practical application.
In a further embodiment, the matrigel coated cell detection module simulates an in vivo environment of brain glioma organoid cells by using matrigel, observes and analyzes proliferation of the brain glioma organoid cells in a three-dimensional space, the matrigel coated cell detection module detects a matrigel simulated brain glioma organoid cell sample dyed by fluorescein PI by adopting a flow cell detection method, the flow cell detection method determines DNA content contained in each cell by detecting fluorescence intensity of the fluorescein PI, the flow cell detection method distinguishes cells in different periods according to a DNA content scatter diagram, determines proportion of cells in different periods in the sample, and cell periods comprise four stages of G1 phase, S phase, G2 phase and M phase, and finally calculates cell proliferation rate and period analysis result according to proportion of cells in each period.
In a further embodiment, the chemiluminescent method detection module employs coupled luciferase staining to achieve selective labeling of apoptotic cells, the apoptotic cells are catalyzed by the coupled luciferase to generate luminescent signals, and the chemiluminescent method detection module uses a fluorescence microscope and a filter to observe and screen emission spectra to obtain high selectivity and sensitivity of apoptotic cells, thereby achieving detection of apoptosis.
In a further embodiment, the ATP triphosadenine quantitative analysis and detection module mixes trichloroacetic acid with a brain glioma organoid model, the mixed solution of trichloroacetic acid and the brain glioma organoid model breaks tissue cells in the brain glioma organoid model through ultrasonic mechanical breaking and releases ATP triphosadenine, the ATP triphosadenine quantitative analysis and detection module uses a centrifuge, an ATPIase inhibitor and a chloroform organic solvent to centrifuge the mixed solution after breaking the tissue, prevents ATP triphosadenine degradation and separates out ATP triphosadenine in supernatant, the ATP triphosadenine quantitative analysis and detection module uses fluorescein, hydrogen peroxide and luciferase catalyst to react with the separated ATP triphosadenine to generate a product capable of emitting fluorescent signals, and finally the ATP quantitative analysis and detection module quantitatively calculates ATP content according to fluorescent intensity through fluorescent signals generated by observation and analysis, thereby realizing the determination of the activity of the brain glioma organoid model and improving the convenience and accuracy of the brain glioma organoid drug sensitivity detection.
In a specific embodiment, the matrigel-coated cell detection module is used for simulating the in-vivo environment of the brain glioma organoid cells by using matrigel and observing and analyzing the growth and migration conditions of the brain glioma organoid cells in a three-dimensional space. Specifically, the module can help researchers to restore the physiological environment in brain tissues more truly, and the behavior characteristics and interaction mechanisms of the glioma organoid cells in three-dimensional space can be known deeply through monitoring parameters such as cell morphology, proliferation rate, migration capacity and the like. The main functions of the module are as follows: providing a controllable and real experimental environment: the internal tissue environment can be simulated through the matrigel, so that the experimental result is closer to the actual situation. Observing and analyzing the growth and migration of glioma organoid cells in three-dimensional space: relevant parameters such as proliferation rate, migration distance, etc. can be quantitatively monitored and recorded, so that the behavior characteristics and interaction mechanism can be deeply understood. Provides a theoretical basis for the treatment and prevention of related diseases: potential therapeutic and prophylactic measures can be explored by understanding the behavioral characteristics of brain glioma organoid cells.
The chemiluminescent method detection module has the function of realizing the selective marking of apoptotic cells by coupling luciferase staining, and further observing and screening an emission spectrum by using a fluorescence microscope and a filter to obtain high selectivity and sensitivity of apoptotic cells, thereby realizing the detection of apoptosis conditions. In particular, the module can help researchers to quickly and accurately detect apoptosis, and has the following advantages: high selectivity: only apoptotic cells are labeled by coupled luciferase staining, thereby improving the selectivity of detection. High sensitivity: after being catalyzed by coupled luciferase, the generated luminescent signal is stronger, and apoptotic cells can be effectively detected at low concentration. Quick and convenient: the fluorescence microscope and the filter are used for observing and screening the emission spectrum, and the method has the characteristics of simplicity in operation, rapidness, convenience and the like. The reliability is high: by using the chemiluminescence method for detection, misjudgment caused by interference of other factors can be avoided, so that the reliability of a detection result is improved. In a word, the chemiluminescent method detection module has the characteristics of high selectivity, high sensitivity, rapidness, convenience, high reliability and the like, can be used for detecting apoptosis conditions, and provides a theoretical basis for diagnosis and treatment of related diseases.
The ATP triphosadenine quantitative analysis detection module has the function of evaluating the activity of the ATP triphosadenine by measuring the ATP content in the brain glioma organoid model and improving the convenience and accuracy of the brain glioma organoid drug sensitivity detection. In particular, the module can help researchers to quickly and accurately determine the ATP content in a sample, thereby achieving the following goals: assessing the activity of a glioma organoid model: ATP is a key substance of intracellular energy metabolism, and the ATP content in glioma organoids models can reflect their metabolic activity level. Thus, by measuring the ATP content in the sample, the activity of the glioma organoid model can be assessed. The convenience and the accuracy of drug sensitivity detection are improved: in drug sensitive assays, it is often necessary to determine the ATP content of a cell or tissue sample to determine the effect of the drug on cellular metabolism. The ATP adenosine triphosphate quantitative analysis detection module can rapidly and accurately complete the step, so that the convenience and the accuracy of drug sensitivity detection are improved. Provides basic data for brain glioma organoid related studies: the ATP adenosine triphosphate quantitative analysis detection module can help researchers to obtain basic data of ATP content in brain glioma organoids, and support further related research. In conclusion, the ATP triphosphates quantitative analysis detection module can be used for evaluating the activity of brain glioma organoids and has important function in drug sensitivity detection. Meanwhile, the module can also provide basic data support for related research.
In a further embodiment, the nuclear magnetic resonance detection method comprises the steps of pretreating a cell culture solution by using a centrifuge, a filtering membrane and a PBS buffer solution, removing bubbles and particles in the cell culture solution, generating specific nuclear magnetic resonance signals by using metabolites and compounds in the cell culture solution through a nuclear magnetic resonance instrument, detecting signals of different molecules in the cell culture solution, performing nuclear magnetic resonance spectrum analysis on the signals of the molecules through the statistics software of the cytoprofiler, determining the proportion of the metabolites and the compounds in a sample, and finally indicating the influence of the selected drugs on the growth or metabolism of the cells according to the proportion of the metabolites and the compounds in the sample.
In particular embodiments, nuclear Magnetic Resonance (NMR) is a common physical detection technique that can be used to detect the structure and dynamic behavior of molecules and atoms. In the biomedical field, nuclear magnetic resonance technology is widely applied to non-invasive detection of biomacromolecules, cells, tissues and the like, and can be used for detecting the activity of cells in culture fluid according to the drug sensitivity detection requirement of brain glioma organoid models. Specifically, nuclear magnetic resonance techniques can be used to detect changes in signal intensity and quantity of metabolites in a sample, and thus infer cellular activity levels and their responses to different drugs. For example, in performing drug sensitivity detection of brain glioma organoid model, different drugs may be added to the culture broth, and samples may be collected separately for nuclear magnetic resonance detection. By comparing the signal intensity and the quantity change of the metabolites in different samples, the influence and the effect of different drugs on the brain glioma organoid model can be evaluated, so that the nuclear magnetic resonance technology is used as a non-invasive, high-sensitivity and high-resolution detection method, can be used for detecting the activity of cells in the culture solution, and can realize the drug sensitivity detection of the brain glioma organoid model.
In a further embodiment, a method for detecting drug sensitivity of a brain glioma organoid is applied to the method for constructing a brain glioma organoid, the method for detecting drug sensitivity comprising the steps of:
transferring a brain glioma organoid culture plate into a biosafety cabinet, transferring the brain glioma organoid in a suction hole of a pipetting gun into a sterile 15mL centrifuge tube, repeatedly blowing, centrifuging in a centrifuge at the temperature of-4 ℃ to 4 ℃, discarding supernatant to leave a precipitate, re-suspending by adopting a culture medium according to the volume of the precipitate, taking an equal volume of cell suspension and trypan blue solution, fully and uniformly mixing the cell suspension and the trypan blue solution according to the ratio of 1:1, counting, and performing drug sensitive plating and culture according to a drug recommendation scheme;
dividing the brain glioma organoid model into a drug treatment group and a control group of temozolomide, methylbenzyl hydrazine, lomustine, vincristine, carmustine, etoposide, irinotecan, cisplatin, carboplatin and bevacizumab;
step three, respectively adding temozolomide, methylbenzyl hydrazine, lomustine, vincristine, carmustine, etoposide, irinotecan, cisplatin, carboplatin and bevacizumab into each drug treatment group, and gradually increasing the concentrations of 0.1g/ml, 0.2g/ml, 0.3g/ml, 0.4g/ml and 0.5g/ml to obtain drug concentration-effect relation data, wherein no drug is added into the control group, and the culture plates of the drug treatment group and the control group are placed into a culture box for culture;
Step four, adding an equivalent amount of ATP luminescent detection reagent into culture plate holes of each drug treatment group and each control group, detecting by adopting an enzyme-labeled instrument with a luminescent detection function, processing and analyzing cell values in each sample by adopting a nuclear magnetic resonance detection method and cell profilometer statistical software, and detecting the influence of drugs on the growth or metabolism of cells;
and fifthly, correspondingly comparing the cell growth or metabolism conditions in the drug treatment group and the control group to finally obtain the drug sensitivity of temozolomide, methylbenzyl hydrazine, lomustine, vincristine, carmustine, etoposide, irinotecan, cisplatin, carboplatin and bevacizumab to brain glioma organoids.
While specific embodiments of the present invention have been described above, it will be understood by those skilled in the art that these specific embodiments are by way of example only, and that various omissions, substitutions, and changes in the form and details of the methods and systems described above may be made by those skilled in the art without departing from the spirit and scope of the invention. For example, it is within the scope of the present invention to combine the above-described method steps to perform substantially the same function in substantially the same way to achieve substantially the same result. Accordingly, the scope of the invention is limited only by the following claims.
Claims (10)
1. A brain glioma organoid construction method is characterized in that: the construction method comprises the following steps:
firstly, separating cells from a glioma sample by primary separation of tissue cells, and inoculating and culturing the cells according to a cell counting result;
step two, planting cells on the three-dimensional biological scaffold by adopting a three-dimensional cell culture method, and culturing the cells into a brain glioma organoid model with a three-dimensional structure, wherein the three-dimensional cell culture method comprises the steps of preparing a scaffold module, a scaffold surface modification module and a cell three-dimensional culture module;
in the second step, the output end of the preparation bracket module is connected with the input end of the bracket surface modification module, and the output end of the bracket surface modification module is connected with the input end of the cell three-dimensional culture module;
step three, performing mechanical property test and corrosion resistance test on the three-dimensional biological scaffold of the brain glioma organoid model by adopting chemical abrasion test, wherein the chemical abrasion test comprises mechanical stress test and chemical corrosion test;
step four, biological characteristics and efficacy of a brain glioma organoid model are evaluated through a chemical and physical detection method, wherein the chemical and physical detection method comprises a matrigel coated cell detection module, a chemiluminescence method detection module and an ATP adenosine triphosphate quantitative analysis detection module;
In the fourth step, the output end of the matrigel coated cell detection module is connected with the input end of the chemiluminescence method detection module, and the output end of the chemiluminescence method detection module is connected with the input end of the ATP adenosine triphosphate quantitative analysis detection module;
fifthly, adding the selected medicine into a culture plate with a brain glioma organoid model, determining the influence of the selected medicine on the growth or metabolism of cells through a nuclear magnetic resonance detection method and a cell profiler statistical software, and realizing the drug sensitivity detection of the brain glioma organoid model, wherein the selected medicine at least comprises temozolomide, methylbenzyl hydrazine, lomustine, vincristine, carmustine, etoposide, irinotecan, cisplatin, carboplatin and bevacizumab.
2. The method for constructing brain glioma organoids according to claim 1, wherein: the preparation stent module adopts matrigel as a three-dimensional biological stent material, the stent surface modification module forms a silicon dioxide layer on the matrigel three-dimensional biological stent through trichloromethyl silane, the mechanical strength of the matrigel three-dimensional biological stent is improved to be 6.5 MPa, and the stent surface modification module forms a pore diameter of 150 on the surface of the matrigel three-dimensional biological stent through ion implantation treatment Is provided to enhance cell adhesion.
3. The method for constructing brain glioma organoids according to claim 1, wherein: the cell three-dimensional culture module is used for enabling cells to invade the inside of the matrigel three-dimensional biological scaffold by immersing the matrigel three-dimensional biological scaffold in a cell culture solution, and controlling the pressure, the temperature and the cell culture solution by adopting a model predictive control algorithmThe concentration is used for realizing accurate control of the three-dimensional cell culture environment, and the working method of the model predictive control algorithm comprises the following steps:
step one, establishing the pressure, the temperature and the culture solution by adopting a state space equationDynamic equations between concentrations, which are used to describe pressure, temperature and +.>The model predictive control algorithm is based on algebraic equation set theory to control the pressure, temperature and +.>The concentration is used as an unknown variable, a culture solution dynamic model is constructed by utilizing a reaction rate constant and a growth rate, and the calculation formula of the dynamic equation is as follows:
（1）
in the case of the formula (1),for dynamic equation>For the pressure of the culture broth, < > is given>For the temperature of the culture solution, < > is->Is>Concentration of- >A coefficient matrix which is a state space equation;
step two, the culture solution dynamic model regards the three-dimensional cell culture environment as a space grid consisting of cells through a system dynamics method, and each cell comprises 1The nutrient, the dynamic model of said culture solution predicts the condition of the future change of pressure, temperature, carbon dioxide of the culture solution on the basis of the interaction relation and cell behavior law among cell, the computational formula of the dynamic model of said culture solution predicts the pressure variation of the culture solution is:
（2）
in the formula (2) of the present invention,predicting the pressure variation of the culture solution for the culture solution dynamic model,/->For cell adhesion between the cells +.>Predicting the current pressure value of the culture solution for the culture solution dynamic model,/>is the cell migration velocity;
the temperature change amount calculation formula of the culture solution dynamic model prediction culture solution is as follows:
（3）
in the formula (3) of the present invention,predicting the temperature variation of the culture solution for the culture solution dynamic model,/->For the heating power of the heater, +.>For cultivation time, ->Heat conduction rate for three-dimensional cell culture environment, +.>The cell three-dimensional culture environment capacity is used for cells;
the calculation formula for predicting the carbon dioxide concentration variation of the culture solution by the culture solution dynamic model is as follows:
（4）
In the formula (4) of the present invention,predicting the carbon dioxide concentration variation of the culture solution for the culture solution dynamic model,/->For cell growth rate, ++>Supplying system power for carbon dioxide, < >>Rate of production of cellular metabolites;
step three, a main controller is adopted to output control signals in real time according to the prediction result of the dynamic model of the culture solution and the target set value of the three-dimensional culture environment of the cells, and the model prediction control algorithm adjusts the power of a heater, the opening of an exhaust valve and a carbon dioxide supply system to enable the pressure, the temperature and the carbon dioxide supply system to be in accordance with the control signalsThe concentration is kept at a target set value, and the calculation formula of the control signal is as follows:
（5）
in the formula (5) of the present invention,for control signal +.>Proportional gain for model predictive control algorithm, +.>Outputting control signal time for the main controller, +.>To limit the saturation clipping of the size range of the output control signal.
4. The method for constructing brain glioma organoids according to claim 1, wherein: the mechanical stress test adopts a hydraulic system to vibrate or load a three-dimensional biological scaffold of a brain glioma organoid model, simulates the service condition of the three-dimensional biological scaffold by mechanical stress in the internal environment of a human body, adopts a PID control algorithm to set the pressure and flow of a hydraulic cylinder according to test requirements, realizes that the hydraulic cylinder converts liquid into mechanical motion to act on a three-dimensional biological scaffold sample of the brain glioma organoid model, acquires test data of the load and deformation of the three-dimensional biological scaffold of the brain glioma organoid model in the test process through a sensor, adopts LabVIEW data analysis software to analyze and process the acquired test data, and adopts a curve fitting function to compare the test data with a theoretical model to obtain the yield strength performance of the three-dimensional biological scaffold.
5. The method for constructing brain glioma organoids according to claim 1, wherein: the chemical corrosion test adopts a sodium carbonate-potassium hydroxide buffer solution to simulate the degradation condition of the three-dimensional biological scaffold in the internal environment of a human body, and after the three-dimensional biological scaffold sample of the glioma organoid model is soaked in the sodium carbonate-potassium hydroxide buffer solution for 7 days, the chemical corrosion test adopts a scanning electron microscope to compare the appearance, the quality and the chemical components of the sample before and after the test, and the corrosion resistance of the three-dimensional biological scaffold of the glioma organoid model in the internal environment of the human body is evaluated.
6. The method for constructing brain glioma organoids according to claim 1, wherein: the matrigel coated cell detection module is used for simulating the in-vivo environment of brain glioma organoid cells, observing and analyzing the proliferation condition of the brain glioma organoid cells in a three-dimensional space, detecting the matrigel coated cell detection module which is used for simulating a brain glioma organoid cell sample dyed by fluorescein PI by adopting a flow cell detection method, determining the DNA content contained in each cell by detecting the fluorescence intensity of the fluorescein PI, distinguishing the cells in different periods according to a DNA content scatter diagram by the flow cell detection method, determining the proportion of the cells in different periods in the sample, wherein the cell period comprises four stages of G1 phase, S phase, G2 phase and M phase, and finally calculating the cell proliferation rate and the period analysis result according to the proportion of the cells in each period.
7. The method for constructing brain glioma organoids according to claim 1, wherein: the chemiluminescence method detection module adopts coupled luciferase to dye to realize selective marking of apoptotic cells, the apoptotic cells generate luminous signals after being catalyzed by the coupled luciferase, and the chemiluminescence method detection module uses a fluorescence microscope and a filter to observe and screen emission spectra, so that high selectivity and sensitivity of the apoptotic cells are obtained, and the apoptosis condition of the apoptotic cells is detected.
8. The method for constructing brain glioma organoids according to claim 1, wherein: the ATP adenosine triphosphate quantitative analysis detection module adopts trichloroacetic acid to mix with a brain glioma organoid model, tissue cells in the brain glioma organoid model are crushed and ATP adenosine triphosphate is released through ultrasonic mechanical crushing, the ATP adenosine triphosphate quantitative analysis detection module adopts a centrifuge, an ATPIase inhibitor and a chloroform organic solvent to centrifuge and prevent ATP adenosine triphosphate degradation and separate ATP adenosine in supernatant, the ATP adenosine triphosphate quantitative analysis detection module adopts a mixed reaction of fluorescein, hydrogen peroxide and luciferase catalyst to generate a product capable of emitting fluorescent signals, and finally the ATP content is quantitatively calculated according to fluorescent intensity by observing and analyzing fluorescent signals generated by a fluorescent detector, so that the activity of the brain glioma organoid model is measured and the convenience and accuracy of the brain glioma organoid drug sensitivity detection are improved.
9. The method for constructing brain glioma organoids according to claim 1, wherein: the nuclear magnetic resonance detection method comprises the steps of pretreating a cell culture solution by using a centrifuge, a filtering membrane and a PBS buffer solution, removing bubbles and particles in the cell culture solution, generating specific nuclear magnetic resonance signals by using metabolites and compounds in the cell culture solution through a nuclear magnetic resonance instrument, detecting signals of different molecules in the cell culture solution, carrying out nuclear magnetic resonance spectrum analysis on the signals of the molecules through statistical software of the cytoprofiler, and determining the proportion of the metabolites and the compounds in a sample, wherein the nuclear magnetic resonance detection method finally indicates the influence of the selected drugs on the growth or metabolism of the cells according to the proportion of the metabolites and the compounds in the sample.
10. A brain glioma organoid drug sensitivity detection method is characterized in that: a brain glioma organoid construction method applied to any one of claims 1 to 9, said drug sensitivity detection method comprising the steps of:
transferring a brain glioma organoid culture plate into a biosafety cabinet, transferring the brain glioma organoid in a suction hole of a pipetting gun into a sterile 15mL centrifuge tube, repeatedly blowing, centrifuging in a centrifuge at the temperature of-4 ℃ to 4 ℃, discarding supernatant to leave a precipitate, re-suspending by adopting a culture medium according to the volume of the precipitate, taking an equal volume of cell suspension and trypan blue solution, fully and uniformly mixing the cell suspension and the trypan blue solution according to the ratio of 1:1, counting, and performing drug sensitive plating and culture according to a drug recommendation scheme;
Dividing the brain glioma organoid model into a drug treatment group and a control group of temozolomide, methylbenzyl hydrazine, lomustine, vincristine, carmustine, etoposide, irinotecan, cisplatin, carboplatin and bevacizumab;
step three, respectively adding temozolomide, methylbenzyl hydrazine, lomustine, vincristine, carmustine, etoposide, irinotecan, cisplatin, carboplatin and bevacizumab into each drug treatment group, and gradually increasing the concentrations of 0.1g/ml, 0.2g/ml, 0.3g/ml, 0.4g/ml and 0.5g/ml to obtain drug concentration-effect relation data, wherein no drug is added into the control group, and the culture plates of the drug treatment group and the control group are placed into a culture box for culture;
step four, adding an equivalent amount of ATP luminescent detection reagent into culture plate holes of each drug treatment group and each control group, detecting by adopting an enzyme-labeled instrument with a luminescent detection function, processing and analyzing cell values in each sample by adopting a nuclear magnetic resonance detection method and cell profilometer statistical software, and detecting the influence of drugs on the growth or metabolism of cells;
and fifthly, correspondingly comparing the cell growth or metabolism conditions in the drug treatment group and the control group to finally obtain the drug sensitivity of temozolomide, methylbenzyl hydrazine, lomustine, vincristine, carmustine, etoposide, irinotecan, cisplatin, carboplatin and bevacizumab to brain glioma organoids.
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